Effects Of SKF Research Articles

Abstract P005: GRK4 Inhibitors Suppress Breast Cancer Cell Growth And Potentiate The Inhibitory Effect Of Dopaminergic D 1 Receptor Agonist SKF38393

The renal dopaminergic D 1 receptor (D 1 R) regulates sodium excretion which is terminated by phosphorylation by G protein-coupled receptor kinases 4 (GRK4). GRK4 gene variants are associated with increased GRK4 activity and reduced sodium excretion resulting in hypertension. Breast cancer incidence is higher in hypertensive women. We found that GRK4 is a potential molecule linking these two diseases. We hypothesized that GRK4 inhibitors would be beneficial to patients with hypertension and breast cancer. Three potential GRK4 inhibitors (compounds A, B, and C) were tested for their effect on the growth of breast cancer cells MDA-MB-468, MCF-7, and benign mammary epithelial cells MCF-10A controls. These cell lines had high, low, and no GRK4 expression respectively. Growth of MDA-MB-468 and MCF-7 cells was effectively inhibited by compound C with IC 50 16.8±1.9 nM (n=2). MCF-10A cells were relatively resistant to compound C with IC 50 49.3±4.0 nM (n=3) that is significantly higher than the cancer cells (p<0.001). Compound B was the least effective inhibitor in all three cell lines (IC 50 was 1.5-2.3 μM). Growth inhibition of compound A was similar in MCF-7 and MCF-10A cells but less effective in MDA-MB-468 cells indicating GRK4 inhibition may not be the only target for growth inhibition of this compound. It has been reported that D 1 R agonists inhibit growth of breast cancer cells. We hypothesized that blockade of GRK4 would increase sensitivity of breast cancer cells to the inhibitory effect of a D 1 R agonist. In MDA-MB-468 cells, SKF38393 (SKF) at 20 μM caused a 36% reduction in cell number (from 276.7±0.47E4 to 177.7±4.33E4). Compound C alone reduced cell number by 37% (172.4±0.04E4, 5 nM) and 51% (136.3±7.87E4, 10 nM) respectively. Combination treatment induced more reduction in cell number, 63% (100.4±5.54E4, 5 nM) and 84% (44.1±12.7E4, 10 nM). Similarly, Compound A also enhanced the inhibitory effect of SKF. A left-shift of the SKF dose-response curve in GKR4 knock-down MDA-MB-468 cells confirmed that inhibition of GRK4 increases sensitivity of breast cancer cells to SKF. Our preliminary results suggest that targeting GRK4 with compound C and a dopaminergic agonist could be a novel strategy for breast cancer therapy especially for the patients with hypertension.

Role of paraventricular hypothalamic dopaminergic D1 receptors in food intake regulation of food-deprived rats

Dopaminergic neurons play an important role on central regulatory mechanisms of feeding behavior. Dopamine receptors are distributed within the hypothalamus and densely localized in the paraventricular hypothalamic nucleus (PVN). From these ideas we postulated that PVN D1 receptors may play a role in regulating the food intake behavioral process. In this paper, we considered the effects of SKF38393, a D1 receptor agonist, and the D1 receptor antagonist (SCH23390), on food intake of conscious rats deprived of food for 24h. Our findings revealed that intraparaventricular injections of SKF383993 (0.3–5µg) stimulated food intake behavior in a dose dependent manner. This stimulatory effect of SKF3833 persisted over 2h of the monitoring period. The PVN injections of D1 receptor antagonist were associated with dose-dependent inhibition of food intake. SCH23390 (0.01µg) was also administered 5min before intraparaventricular injection of SKF3833. The results showed that SCH23390 suppressed stimulated food intake induced by SKF38393 (1.2µg). In conclusion, endogenous dopamine impact PVN D1 receptors and may be a factor in regulating the food intake behavioral process.

Dopamine Receptors Modulate Cytotoxicity of Natural Killer Cells via cAMP-PKA-CREB Signaling Pathway

Dopamine (DA), a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK) cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R) with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R) with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB) level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA), prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC), counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The results may provide more targets of therapeutic strategy for neuroimmune diseases.

Rapid dopaminergic and GABAergic modulation of calcium and voltage transients in dendrites of prefrontal cortex pyramidal neurons

The physiological responses of dendrites to dopaminergic inputs are poorly understood and controversial. We applied dopamine on one dendritic branch while simultaneously monitoring action potentials (APs) from multiple dendrites using either calcium-sensitive dye, voltage-sensitive dye or both. Dopaminergic suppression of dendritic calcium transients was rapid (<0.5 s) and restricted to the site of dopamine application. Voltage waveforms of backpropagating APs were minimally altered in the same dendrites where dopamine was confirmed to cause large suppression of calcium signals, as determined by dual voltage and calcium imaging. The dopamine effects on dendritic calcium transients were fully mimicked by D1 agonists, partially reduced by D1 antagonist and completely insensitive to protein kinase blockade; consistent with a membrane delimited mechanism. This dopamine effect was unaltered in the presence of L-, R- and T-type calcium channel blockers. The somatic excitability (i.e. AP firing) was not affected by strong dopaminergic stimulation of dendrites. Dopamine and GABA were then sequentially applied on the same dendrite. In contrast to dopamine, the pulses of GABA prohibited AP backpropagation distally from the application site, even in neurons with natural Cl− concentration (patch pipette removed). Thus, the neocortex employs at least two distinct mechanisms (dopamine and GABA) for rapid modulation of dendritic calcium influx. The spatio-temporal pattern of dendritic calcium suppression described in this paper is expected to occur during phasic dopaminergic signalling, when midbrain dopaminergic neurons generate a transient (0.5 s) burst of APs in response to a salient event.

Repeated exposure to MDMA and amphetamine: sensitization, cross-sensitization, and response to dopamine D1- and D2-like agonists

Acute exposure to (±) 3, 4-methylenedioxymethamphetamine (MDMA) produces hyperlocomotion that is preferentially expressed in the periphery of closed chambers. Following repeated administration, however, a sensitized hyperlocomotor response is preferentially expressed in the center of an activity box, so that the response resembles the more generalized activity that is produced by D-amphetamine (AMPH). The present study was designed to determine whether common neuroadaptations underlie the acute and sensitized responses to MDMA and AMPH. Rats were pretreated with five daily injections of MDMA (10.0 mg/kg), AMPH (2.0 mg/kg), or saline. Following a 2-day drug-free period, dose-response curves for hyperactivity produced by MDMA (2.5-10.0 mg/kg), AMPH (0.5-2.0 mg/kg), SKF-81297 (1.0-2.0 mg/kg), or quinpirole (0.25-1.0 mg/kg) were obtained. Effects of MDMA and AMPH were increased by pretreatment with both drugs. The sensitized response following MDMA exposure was preferentially expressed in the center compartment, but, following AMPH pretreatment, the sensitized response was observed in both compartments. Cross-sensitization was unidirectional; AMPH pretreatment failed to sensitize to the effects of MDMA, but MDMA pretreatment sensitized to the effects of AMPH. MDMA and AMPH pretreatment produced marginal increases in the effects of SKF-81297. The response to quinpirole was, however, greater following MDMA, but not AMPH, pretreatment. These data suggest that repeated MDMA exposure produces sensitization via a unique neurochemical effect.

Dopamine D 1-like receptors play only a minor role in the increase of striatal dopamine induced by striatally applied SKF38393

We studied the effects of the intra-striatal infusion of Ca 2+-free medium on the intra-striatal injection of 0.5 μg SKF38393-induced striatal dopamine efflux. It is discussed that the amount of extracellular, striatal dopamine seen after striatally applied SKF38393, is the overall result of the (a) release of dopamine from the alpha-methyl-para-tyrosine-sensitive and Ca 2+-insensitive pool of newly synthesised dopamine, (b) release of dopamine from the reserpine-sensitive and Ca 2+-sensitive storage pool, (c) inhibition of uptake of dopamine into nerve terminals and glial cells, and (d) facilitation respectively of the inhibition of uptake into blood vessels: dopamine D 1-like receptors play only a very limited role in these processes. The present study underlines our previous notion that the effects of SKF38393 cannot simply be ascribed to the dopamine D 1-like receptor stimulation (Saigusa et al., 2009): in fact, the present study clearly reveals that SKF38393 is not at all selective in that respect.

Contribution of vesicular and cytosolic dopamine to the increased striatal dopamine efflux elicited by intrastriatal injection of SKF38393

Like dexamphetamine, SKF38393 induces an increase in striatal dopamine efflux which is insensitive for tetrodotoxin, Ca 2+ independent and prevented by a dopamine transporter inhibitor. The dexamphetamine-induced striatal dopamine efflux originates from both the reserpine-sensitive vesicular dopamine pool and the alpha-methyl-para-tyrosine-sensitive cytosolic dopamine pool. Given the similarities between dexamphetamine and SKF38393, we hypothesized that both types of pool also contribute to the striatally applied SKF38393-induced dopamine efflux. Using in vivo microdialysis technique, we analysed the contribution of these pools to the SKF38393-induced striatal dopamine efflux in freely moving rats. The increase of dopamine efflux induced by 1.5 μg SKF38393 was largely prevented by either reserpine (5 mg/kg i.p., given 24 h earlier) or alpha-methyl-para-tyrosine (250 mg/kg i.p., given 2 h earlier), showing that both the vesicular dopamine pool and the cytosolic dopamine pool contribute to the SKF38393-induced increase in striatal dopamine efflux. The sum of the amounts of dopamine that was sensitive to either reserpine or alpha-methyl-para-tyrosine, was greater than 100%, namely 137.6% of the basal dopamine level and 143.9% of the SKF38393-induced dopamine level, suggesting that striatally applied SKF38393 promotes the redistribution of dopamine from vesicles to the cytosol, and vice versa. The finding that the combined treatment of reserpine and alpha-methyl-para-tyrosine only inhibited the SKF38393-induced striatal dopamine efflux till 86.0% of the control, is ascribed to the notion that SKF38393 can also inhibit the re-uptake of dopamine. The latter conclusion has far-reaching consequences for studies in which the effects of SKF38393 are simply ascribed to its dopamine D 1 receptor stimulation capacity.

Effects of SKF-38393, a dopamine D 1 receptor agonist on expression of amphetamine-induced behavioral sensitization and expression of immediate early gene arc in prefrontal cortex of rats

Repeated administrations of psychostimulants into rodents produce behavioral sensitization. We examined whether a dopamine D 1 agonist can reverse behavioral sensitization once established by repeated amphetamine (AMP) administrations and determined the mRNA expression levels of the D 1 and D 2 receptors, metabotropic glutamate receptor 1 (mGluR1), and activity-regulated cytoskeleton-associated protein (arc) in rats. Rats were pretreated with six intermittent AMP injections. Following a 14-day withdrawal period, the rats were divided into six groups and treated with either SKF-38393 (SKF; dopamine D 1 agonist), SCH-23390 (SCH; selective D 1 antagonist), YM-09151-2 (YM; selective D 2 antagonist), SKF + SCH, SKF + YM or physiological saline once daily for 5 days. Three days or 4 weeks after the reversal treatments, all the rats were rechallenged with AMP. D 1 and D 2 antagonist treatments produced no significant decreases in locomotor activity or stereotyped behavior rate, respectively. In the SKF treatment group, stereotyped behavior rate decreased markedly after the three-day and four-week withdrawal periods. SKF + SCH treatment inhibited the effect of SKF treatment. The rats in the other groups that received AMP with or without SKF were decapitated 1 h after treatment, and the mRNA levels of the D 1 and D 2 receptors, mGluR1, and arc were measured by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR). AMP administration significantly increased arc level. SKF also increased arc level significantly after the first single injection and after repeated injections of AMP during the pretreatment. There was no significant difference in arc expression level between the saline and SKF treatment groups after the AMP challenge, suggesting that arc expression level is not involved in the reversal effects of SKF in AMP sensitization.

Lesion of Subthalamic Nucleus in Parkinsonian Rats : Effects of Dopamine D1and D2Receptor Agonists on the Neuronal Activities of the Substantia Nigra Pars Reticulata

It was hypothesized that dopamine agonist administration and subthalamic nucleus (STN) lesion in the rat might have a synergistic effect on the neuronal activities of substantia nigra pars reticulata (SNpr) as observed in patients with Parkinson's disease. The effects of SKF38393 (a D(1) receptor agonist) and Quinpirole (a D(2) receptor agonist) were compared in parkinsonian rat models with 6- hydroxydopamine (6-OHDA) after STN lesion. SKF38393 and Quinpirole were consecutively injected intrastriatally. SNpr was microrecorded to ascertain the activity of the basal ganglia output structure. The effect of SKF38393 or Quinpirole injection on the firing rate and firing patterns of SNpr was investigated in medial forebrain bundle (MFB) lesioned rats and in MFB+STN lesioned rats. The administration of SKF38393 decreased SNpr neuronal firing rates and the percentage of burst neurons in the MFB lesioned rats, but did not alter them in MFB+STN lesioned rats. The administration ofQuinpirole significantly decreased the spontaneous firing rate in the MFB lesioned rats. However, after an additional STN lesion, it increased the percentage of burst neurons. This study demonstrated that dopamine agonists and STN lesion decreased the hyperactive firing rate and the percentage of burst neurons of SNpr neurons in 6-OHDA lesioned rats, respectively. Quinpirole with STN lesion increased a percentage of burst neurons. To clear the exact interactive mechanism of D(1) and D(2) agonist and the corresponding location, it should be followed a study using a nonselective dopamine agonist and D(1), D(2) selective antagonist.

The α1-adrenergic receptor not the DA1-dopaminergic receptor mediates cyclosporine–SKF38393 renovascular interaction

In this study, we investigated the effect of acute exposure to cyclosporine A (CyA) on renal vasodilations evoked by the DA(1) dopaminergic agonist SKF38393 and whether dopamine DA(1) receptors are directly involved in the interaction. Changes evoked by CyA in SKF38393 vasodilations were evaluated in phenylephrine-preconstricted isolated perfused rat kidneys in the absence and presence of SCH23390, a DA(1) receptor antagonist. SKF38393 (3 x 10(-8) to 3 x 10(-6) mol) produced dose-dependent reductions in the renal perfusion pressure that were significantly attenuated in tissues pretreated with SCH23390 or CyA. Unlike SKF38393, the vasodilatory action of sodium nitroprusside, a nitrovasodilator, was not altered by CyA. The attenuating effect of CyA on SKF38393 vasodilations was preserved in preparations pretreated with SCH23390, suggesting that sites other than DA(1) receptors may be involved in CyA-SKF38393 interaction. The study was then extended to investigate the possible involvement of renal alpha1-adrenoceptors in the interaction. Blockade of alpha(1)-adrenoceptors by prazosin (30 nmol/L) significantly reduced the vasodilatory effect of SKF38393 and virtually abolished the CyA-induced attenuation of SKF38393 responses. Further, CyA failed to alter SKF38393 vasodilations when the renal tone was raised with prostaglandin F2alpha (PGF2alpha), a vasoconstrictor whose effect is independent of alpha(1)-adenoceptors. Together, these findings support earlier reports that both DA(1) and alpha(1)-receptors mediate the renal vasodilatory action of SKF38393 and suggest that CyA interacts selectively with the alpha(1)-receptor component to compromise SKF38393 responses.

Physicochemical Modulation of Agonist-Induced [35S]GTPγS Binding: Implications for Coexistence of Multiple Functional Conformations of Dopamine D1-Like Receptors

Dopamine agonist-stimulated [35S]GTPγS binding to membrane G proteins was studied in select brain regions under experimental conditions that permit the activation of receptor coupling to the G proteins Gi, Gs, or Gq. Agents studied were agonists known to be effective at various dopamine receptor/effector systems and included quinelorane (D2-like/Gi), SKF38393 (D1-like/Gq, D1-like/Gs), SKF85174 (D1-like/Gs), and SKF83959 (D1-like/Gq). Dopamine and SKF38393 significantly stimulated [35S]GTPγS binding to normal striatal membranes by 161% and 67% above controls. Deoxycholate, which enhances agonist-induced phospholipase C (PLC) stimulation, markedly enhanced the agonistic effects of dopamine and SKF38393 to 530% and 637% above controls, respectively. The enhancing effects of deoxycholate were reversed if it was washed off the membranes before agonist addition. The thiol-reducing agent, dithiothreitol, completely abolished the effects of SKF38393 and SKF83959, whereas SKF85174 effects were augmented. Agonist responses were concentration-related, and highest efficacies were obtained in the hippocampus, thus paralleling both the brain regional distribution and agonist efficacies previously observed in phosphoinositide hydrolysis assays. These findings suggest that D1-like receptor conformations that mediate agonist stimulation of Gs/adenylylcyclase may be structurally different from those that mediate Gq/PLC activation. Although the exact mechanism of deoxycholate's effect awaits elucidation, the results are consistent with the emerging concept of functional selectivity whereby deoxycholate could create a membrane environment that facilitates the transformation of the receptor from a conformation that activates Gs/adenylylcyclase to one that favors Gq/PLC signaling.

Effects of dopamine D1 receptor full agonists in rats trained to discriminate SKF 38393

Although the dopaminergic pharmacology of the D1 receptor full agonists, dinapsoline, dihydrexidine and the prodrug ABT-431 have been studied, no information is available on the ability of these agonists to substitute for the D1 agonist SKF 38393 in rats trained to discriminate this compound from vehicle. The present study was designed to characterize the potential D1 discriminative stimulus effects of these compounds. The selective dopamine D1-receptor agonists dihydrexidine [(+/-)-trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a] phenanthridine hydrochloride], ABT-431 [(-)-trans-9,10-diacetyloxy-2-propyl-4,5,5a,6,7,11b-hexahydro-3-thia-5-azacyclopent-1-ena[c]phenanthrene hydrochloride], the diacetyl prodrug derivative of A-86929, and dinapsoline [9-dihydroxy-2,3,7,11b-tetrahydro-1H-naph[1,2,3-de]isoquinoline] were studied in rats trained to discriminate racemic SKF 38393 [(+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol], a selective D1 receptor partial agonist from vehicle. All of the agonists substituted fully for the discriminative stimulus effects of SKF 38393. The rank order of potency for substitution was ABT-431 > dinapsoline > dihydrexidine > SKF 38393. The D1 receptor antagonist, SCH 23390, blocked the discriminative stimulus effects of SKF 38393. The D3/D2-receptor agonist PD 128,907 [S(+)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]-benzopyrano[4,3-b]-1,4-oxazin-9-ol] did not substitute up to doses that produced profound rate-suppressant effects. Thus, consistent with their D1 receptor pharmacology, the full D1-receptor agonists substituted completely for the discriminative stimulus of SKF 38393.

P.6.008 An ethopharmacological assessment of the effects of SKF 10047, a sigma-1 selective agonist, on social interactions between male mice

P.6.008 An ethopharmacological assessment of the effects of SKF 10047, a sigma-1 selective agonist, on social interactions between male mice

D1 and D2 receptor-mediated dopaminergic modulation of visual responses in cat dorsal lateral geniculate nucleus.

The modulatory effects of dopamine (DA) on the visual responses of relay cells of the dorsal aspect of cat lateral geniculate nucleus (dLGN) were tested using local micro-iontophoretic application of DA and application of the receptor-specific agonists SKF38393 (SKF, D1/D5) and quinpirole (QUIN, D2/D3/D4) in the anaesthetized alcuronium-treated cat. The effects of DA and QUIN were clearly dose-dependent: small amounts caused a weak and transient facilitation of visual activity (10-30% increase) preferentially in Y-type relay cells, which changed to a moderate reduction of visual responses when the dose was increased (50%, maximal 70%). The effect of SKF was mainly suppressive and increased with the amount of drug applied (up to 90% reduction). The selective antagonists SCH23390 (SCH, D1) and sulpiride (SULP, D2) reduced the effects of co-applied DA agonists. We found little evidence for a specific dopaminergic modulation of the surround inhibition (stimulus-driven lateral inhibition) although DA slightly facilitated the transmission of weak signals (small stimuli). Nevertheless, some dopaminergic effects seem to be mediated via inhibitory interneurons regulating the strength of sustained or recurrent inhibition. Application of DA agonists during blockade of GABA(A) receptors indicates a direct suppression of relay cells via D1 receptors, an excitation of relay cells via D2 receptors and--with increasing amounts of D2 agonist--probably also an excitation of inhibitory interneurons, which results in an indirect inhibition of dLGN relay cells (predominantly of the X-type). The results are discussed in relation to the impairment of visual functions in Parkinson's disease.

Blockade or stimulation of D1 dopamine receptors attenuates cue reinstatement of extinguished cocaine-seeking behavior in rats.

D(1) dopamine receptor antagonists and agonists attenuate cocaine reinstatement of cocaine-seeking behavior (i.e., responding in the absence of cocaine reinforcement). The present study investigated the effects of a D(1) antagonist (SCH-23390), partial agonist (SKF-38393), and full agonist (SKF-81297) on reinstatement of cocaine-seeking behavior elicited by presentation of cocaine-paired cues. Rats that had been trained to self-administer cocaine with a light/tone stimulus complex paired with each infusion underwent extinction across days. After responding diminished, rats were given response-contingent access to the cocaine-paired stimulus complex. The effects of SCH-23390 (0-10.0 microg/kg), SKF-38393 (0-3.0 mg/kg), and SKF-81297 (0-3.0 mg/kg) on cue reinstatement of cocaine-seeking behavior were examined. The ability of the two D(1) agonists to independently reinstate cocaine-seeking behavior and the effects of SKF-81297 on cocaine reinstatement were also examined. To investigate the possibility of behavioral interference, the effects of SKF-38393 and SKF-81297 on grooming and stereotypy were assessed. SCH-23390 and SKF-81297, but not SKF-38393, attenuated cue reinstatement. However, while SKF-81297 dose-dependently increased response latency, SCH-23390 did not. SKF-81297 also independently reinstated responding at the two lowest doses tested while SKF-38393 had no effect. Furthermore, SKF-81297 decreased cocaine reinstatement and increased response latency only at the highest dose. Finally, stereotypy was observed at all doses of SKF-81297 that also decreased responding, although the patterns of changes in these behaviors did not completely correspond. While the antagonist and full agonist produced similar effects on cocaine-seeking behavior, only the agonist increased response latency, suggesting that different processes mediate the effects of these drugs.

Changes of contrast gain in cat dorsal lateral geniculate nucleus by dopamine receptor agonists

The modulatory effects of dopamine (DA) on the contrast gain of retino-geniculate transmission were tested with local micro-iontophoretical application of DA and the DA receptor agonists SKF38393 (SKF, D1/D5) and quinpirole (QUIN, D2/D3/D4) while recording visually induced spike activity of relay cells of the dorsal aspect of cat lateral geniculate nucleus (dLGN) in the anesthetised and paralyzed preparation. DA and QUIN could either facilitate or inhibit visual activity in a dose-dependent fashion: small amounts caused a facilitation while larger quantities resulted in a more (DA) or less (QUIN) strong inhibition. The effect of SKF was almost always suppressive and increased with the amount of drug applied. The absolute change in activity was depending on stimulus contrast and the strength of the elicited response: facilitation and inhibition of activity was proportional to stimulus contrast and response strength and thus resulted in a changed contrast gain. The results indicate that the visual deficits found in Parkinson's disease patients my be not solely related to retinal dysfunctions.

Opposite roles of D1 and D5 dopamine receptors in locomotion revealed by selective antisense oligonucleotides.

Contralateral rotations induced by the D1-like agonist SKF 38393 in unilaterally 6-hydroxydopamine-lesioned rats were completely prevented by the administration of the D1-like antagonist SCH 23390. A similar result was obtained after intracerebroventricular administration of an antisense oligodeoxynucleotide for the D1 receptor (D1R-as). Contrariwise, administration of a D5R-as potentiated the effects of SKF 38393, showing a 60% increase in the rotational scores. Both effects were reversible upon cessation of D1R-as or D5R-as treatment and were also specific since rotational scores in rats treated with vehicle or with a randomly designed oligodeoxynucleotide were not modified. These results suggest that whereas D1 receptors play a facilitatory role in locomotion, D5 receptors exert an inhibitory effect.

Dopamine stimulation of cardiac β‐adrenoceptors: the involvement of sympathetic amine transporters and the effect of SKF38393

1. Mechanisms underlying beta-adrenoceptor stimulation by dopamine were examined on guinea-pig Langendorff-perfused hearts and isolated cells from the right atrium, by using the chronotropic effects and the enhancement of L-type Ca2+ current (ICa,L) in the presence of prazosin as indicators of beta-adrenoceptor stimulation. Dopamine-induced overflow of noradrenaline (NA) concentrations was measured by high-performance liquid chromatography. 2. Dopamine caused positive chronotropic effects with an EC50 of 2.5 microM and induced NA overflow with a similar EC50 (1.3 microM). The chronotropic effect of dopamine was abolished by bisoprolol (1 microM). 3. The effects of dopamine were maintained during prolonged application, whereas the effects of tyramine faded with time. Dopamine (3 microM) restored the chronotropic effects and the NA release suppressed by pretreatment with tyramine, suggesting a de novo synthesis of NA during the exposure to dopamine. 4. Dopamine (3 microM)-induced NA release was not affected by tetrodotoxin, omega-conotoxin, rauwolscine, ICI118551 or sulpiride, but was inhibited by desipramine, a NA uptake inhibitor (IC50 approximately 1 microM). It was also not affected by GBR12909 and bupropion, dopamine uptake inhibitors in the central nervous system. 5. SKF38393, a D1 receptor partial agonist, potently inhibited the 3 microM dopamine-induced release of NA (IC50 approximately 0.1 microM). D1 receptors are not involved in the DA-induced release of NA, since SCH23390 (3 microM), a potent D1 antagonist, inhibited the NA release only slightly, and dihydrexidine (1 microM) and chloro-APB (1 microM), full D1 agonists, caused no significant NA release. 6. SKF38393 inhibited tyramine-induced overflow of NA, and potentiated the field stimulation-induced NA release. SKF38393 and desipramine retarded the decay of the stimulation-induced tachycardia in a similar manner. These results indicate that SKF38393 is a potent monoamine transport inhibitor and a useful tool for the functional evaluation of indirectly-acting sympathomimetic agonists in the heart. In the presence of SKF38393 (10 microM), dopamine at 1 microM showed no chronotropic effect. 7. Voltage clamp experiments with isolated atrial cells revealed that dopamine is a weak partial agonist. The EC50 for ICa,L stimulation by dopamine was high (13 microM). As a result, dopamine at 1 microM did not affect ICa,L. Bisoprolol abolished the stimulation of ICa,L by dopamine (30 microM), and dihydrexidine (1 microM) did not affect ICa,L. 8. It was concluded that the cardiac effects of dopamine at clinically relevant concentrations (< 1 microM) result almost exclusively from the indirect effect of beta adrenoceptor stimulation, involving the release of NA from sympathetic nerve terminals. The roles of the direct stimulation of beta adrenoceptors by dopamine at these concentrations and the stimulation of postjunctional D1 receptors seem negligible. The desipramine- and SKF38393-sensitive monoamine transporter mediates the release of NA.

D1 receptors mediate dopamine stimulation of immunoreactive atrial natriuretic factor (ANF) release and pro-ANF messenger ribonucleic acid expression of rat hypothalamic neurons in culture.

Atrial natriuretic factor (ANF) and its smaller congeners are produced and released from rat brains to regulate the cardiovascular system, drinking behavior, and neurohormone release at the central level. In the hypothalamus, the adenylyl cyclase-cAMP system plays an important role in modulating the function of ANF neurons in the paraventricular nuclei and the periventricular regions that receive rich dopaminergic inputs. We report here a novel observation of a dopamine (DA) D1 agonist, SKF-38393, modulating immunoreactive (ir) ANF and pro-ANF messenger RNA (mRNA) expression in rat hypothalamic neurons. In long term primary cultures of neonatal rat hypothalamic cells, treatment with SKF-38393 increased irANF secretion in a time-related and dose-dependent manner, with an ED50 of approximately 10(-7) M and a concentration producing maximum effect of 10(-5) M. This stimulating effect of SKF-38393 was mimicked by 10(-5) M DA, a physiological ligand for D1 receptors. Furthermore, both of the stimulatory effects were abolished by SCH-23390, a D1 antagonist. These immunoassay findings were accompanied by corresponding changes in the abundance of pro-ANF mRNA in the cultures, as examined by colorimetric Northern blot analysis. By combining the techniques of in situ hybridization and immunocytochemistry, the mRNA of D1 receptor was colocalized in approximately 90% of the irANF-positive cells in the cultures. In addition, cholera toxin, an irreversible activator of adenylyl cyclases, markedly increased irANF secretion and cAMP production in a dose-dependent manner. This effect mimicked that of D1 agonist-stimulated ANF release, in that the latter also concurrently enhanced cAMP production in the hypothalamic cultures. Furthermore, the stimulatory effect of D1 agonist on irANF release was markedly suppressed by rp-cAMPS, a cAMP antagonist. We, thus, conclude that the release and gene expression of ANF in rat hypothalamic neurons are directly stimulated by DA acting through its D1 receptors on ANF neurons; this effect may operate at the genomic level and is mediated at least in part through activation of the cAMP-dependent kinase-A pathway.

Altered activity patterns following neonatal 6-hydroxydopamine lesions to dopaminergic neurons: effect of SKF-38393

The effects of neonatal dopamine depletion on activity levels and locomotor patterns were tested in rats at 4 months of age. Measurements were taken using an activity pattern monitor. The lesioned group was significantly hypoactive in comparison to the control animals during the initial exposures to the monitor. Examination of locomotor paths indicated that the lesioned animals failed to develop the increasingly organized spatial behavior observed in control animals. In a second study, changes in motility and locomotor patterns before, during, and after the repeated administration of a D 1-dopamine agonist, SKF-38393 (3.0 mg/kg) were investigated. A priming effect, leading to increases in ambulations and fine movements, was observed only in the lesioned animals after repeated administration of SKF-38393. The D 1-dopamine agonist did not change the activity of the unlesioned animals and did not lead to persistent effects in lesioned animals tested 2 and 4 days following the final drug administration. Across the duration of the second study, the lesioned group did not demonstrate hypoactivity. Overall, the experiments showed that neonatal dopamine depletion disrupts the normal patterns of behavior associated with habituation to a novel environment, and repeated administration of SKF-38393 can further alter these abnormal activity patterns.